Rapid cross-border emergence of NDM-5-producing Escherichia coli in the European Union/European Economic Area, 2012 to June 2022

Whole genome sequencing data of 874 Escherichia coli isolates carrying bla NDM-5 from 13 European Union/European Economic Area countries between 2012 and June 2022 showed the predominance of sequence types ST167, ST405, ST410, ST361 and ST648, and an increasing frequency of detection. Nearly a third (30.6%) of these isolates were associated with infections and more than half (58.2%) were predicted to be multidrug-resistant. Further spread of E. coli carrying bla NDM-5 would leave limited treatment options for serious E. coli infections.

Whole genome sequencing data of 874 Escherichia coli isolates carrying bla NDM-5 from 13 European Union/ European Economic Area countries between 2012 and June 2022 showed the predominance of sequence types ST167, ST405, ST410, ST361 and ST648, and an increasing frequency of detection. Nearly a third (30.6%) of these isolates were associated with infections and more than half (58.2%) were predicted to be multidrug-resistant. Further spread of E. coli carrying bla NDM-5 would leave limited treatment options for serious E. coli infections.
Preliminary unpublished results of a survey of carbapenem-and/or colistin-resistant Enterobacterales (CCRE survey) [1] in 36 European countries showed that New Delhi metallo-β-lactamase (NDM)-5 was the most frequently reported carbapenemase in Escherichia coli with detection of bla  in 62 (30.8%) of 201 carbapenemase-producing E. coli isolates collected in 2019. These 62 E. coli isolates were detected in 15 countries and involved various E. coli sequence types (STs), some of which are described as high-risk STs for extraintestinal infections [2]. These findings were of concern and warranted further investigation with the aim to confirm the extent of spread and describe the epidemiological and microbiological characteristics of the detected isolates.

Data collection and analysis
The European Centre for Disease Prevention and Control requested, via its EpiPulse platform, whole genome sequencing (WGS) and epidemiological data on E. coli carrying bla NDM-5 from European Union (EU)/ European Economic Area (EEA) countries. In reply, data for 905 E. coli isolates were received from 13 countries. Twenty-nine isolates did not fulfil the quality criteria (assembled genome 3.7-6.4 Mbp with ≥ 90% core genome loci covered using the EnteroBase core genome multilocus sequence typing scheme (cgMLST [3]), and two isolates did not carry bla NDM-5 . Therefore, data from 874 isolates from the period 2012 to June 2022 were included in the analysis (Table); these isolates are hereafter referred to as being from 'national collections'. The data for this study were deposited in the European Nucleotide Archive under accession numbers PRJEB27363, PRJEB36710, PRJEB45009, PRJEB56146, PRJEB61153 and PRJNA845120.
We downloaded WGS data from the National Center for Biotechnology Information (NCBI) Pathogen Detection on 20 July 2022 and included an additional 2,561 E. coli isolates carrying bla NDM-5 after quality control using the same criteria as described above. We determined the STs with the MLST 2.0 tool from the Danish Technical University (DTU) (version 2.0.9, database version 26 July 2022, using the Achtman MLST scheme for E. coli) [4]. In line with previously published definitions [5], major STs were defined as representing > 10% and minor STs as 5-10% of the isolates from national collections. Resistance genes were determined using E. coli analysis plugin of BioNumerics 7.6.3 (Applied Maths NV/bioMérieux, Sint-Martens-Latem, Belgium) with thresholds of ≥ 90% sequence identity and ≥ 60% sequence length for gene coverage. Fluoroquinolone resistance point mutations were detected using the PointFinder tool from DTU (reference sequences collected on 29 September 2022) [6]. Clusters of related isolates were determined with a threshold of 10 allelic differences based on previously published studies [7] and using the EnteroBase cgMLST scheme [3].
The epidemiological variables collected for isolates from national collections included year and month of sample collection, the location of the healthcare institution submitting the sample (city and National Territorial Units for Statistics level 2 region), the sample type (screening or clinical sample) and site of sampling for clinical samples, the clinical relevance (infection or carriage), the status of the patient (inpatient or outpatient), the type of acquisition (community-or healthcare-associated), age and sex of the patient, travel/ hospitalisation and country of travel/hospitalisation in the 6 months before sampling and an epidemiological link to another patient.

Distribution of sequence types
The overall dataset included 3,435 E. coli isolates carrying bla NDM-5 belonging to 267 STs. Of these, 83 STs were present in the 874 isolates from national collections, with five predominant STs: ST167 (n = 200; 22.9%), ST405 (n = 115; 13.2%), ST410 (n = 96; 11.0%), ST361 (n = 70; 8.0%) and ST648 (n = 65; 7.4%) ( Figure 1). Based on the national collections and the definitions outlined above, ST167, ST405 and ST410 were classified as major STs and ST361 and ST648 as minor STs, and they were grouped together as dominant STs for further analyses. The five dominant STs were detected in all countries that submitted more than 20 isolates, i.e. Denmark, Finland, France, Ireland, the Netherlands and Sweden (Table). The number of detected E. coli isolates carrying bla NDM-5 increased over time ( Figure 2). Isolates from national collections (n = 500) were involved in 162 clusters of which 114 were multi-country clusters.

Discussion
Our results confirmed the emergence of E. coli isolates carrying bla NDM-5 in the EU/EEA initially detected in the CCRE survey and showed the predominance of five STs (ST167, ST410, ST405, ST361 and ST648) known as highrisk clones based on their rapid global spread and their association with virulence and multidrug resistance [8][9][10][11][12][13][14][15][16]. While these STs carrying bla NDM-5 have previously been described in the EU/EEA, reports were limited to a single ST in one [8,9] or two [10] institutions, or to the description of the national epidemiology of one single ST [11] or several STs [12,13]. Only one study including 33 isolates of E. coli carrying bla NDM-5 has previously reported cross-border emergence in Germany and Switzerland [14]. Our study included WGS data on 874 isolates from national reference laboratories in 13 countries and thus provides a more comprehensive analysis of the spread of these high-risk STs in Europe. Of the isolates with available information on prior travel and/ or hospitalisation, 84.2% had a link with a country outside of the EU/EEA, mainly on the African and Asian continents, suggesting potential acquisition outside of the EU/EEA. About 30% of the E. coli isolates carrying bla NDM-5 were documented as associated with infections, emphasising the clinical relevance and the need for early detection. Sixty-two per cent of isolates carried ESBL genes conferring resistance to aztreonam which is not inactivated by metallo-β-lactamases. Together with predicted resistance to aminoglycosides and fluoroquinolones, this leaves very limited options for the treatment of serious E. coli infections. With respect to aminoglycosides, the rmtB-encoded 16S rRNA methylase conferring resistance to novel aminoglycosides is a particular concern [12,17]. Moreover, studies have shown that isolates (over)-producing NDM-5 had increased minimum inhibitory concentrations to cefiderocol, one of the last treatment options for metalloβ-lactamase-producing Enterobacterales [18,19].
A limitation of this study is that it is not based on a standardised sampling protocol but on regular national surveillance with differences in sample collection protocols, national coverage and data completeness and that it is subject to detection and reporting bias. However, the frequent detection in 13 countries indicates that E. coli carrying bla NDM-5 is now established in the EU/EEA. The proportion of carbapenem resistance in E. coli in the EU/EEA has so far been very low, i.e. only 0.2% of invasive isolates [20]. Further spread of E. coli isolates carrying bla NDM-5 in community and healthcare settings, and within and between countries, is likely to increase dissemination with resulting higher levels of carbapenem and multidrug resistance in E. coli infections.

Conclusion
The spread of E. coli carrying bla NDM-5 is occurring rapidly and on a large geographical scale with a considerable risk for increasing carbapenem resistance in E. coli in the EU/EEA within a few years. Early detection and control are required to mitigate adverse consequences for patients and healthcare systems.

Ethical statement
All data were anonymised and collected in accordance with the European Parliament and Council decisions on the epidemiological surveillance and control of communicable disease in the European Community. Ethical approval and informed consent were thus not required.

Data availability
The whole genome sequencing data for this study were deposited in the European Nucleotide Archive under accession numbers PRJEB27363, PRJEB36710, PRJEB45009, PRJEB56146, PRJEB61153 and PRJNA845120.

Conflict of interest
None declared.

Figure 3
Distribution of Escherichia coli isolates with bla NDM-5 co-carrying additional carbapenemase (n = 180) or ESBL (n =  AKP, MC, VM, JOI, MPV, KW, BM: compilation and analysis of national data, interpretation of results; DP, DLM: design of study and interpretation of results; AK: design and coordination of study, epidemiological analysis, and drafting of manuscript. All authors: review of manuscript.